Acute myeloid leukemia (AML) is a very aggressive malignancy with few long-term survivors. New treatment strategies are urgently needed to improve the survivorship of patients with this disease. The control of protein turnover is frequently disrupted in cancer and the mechanistic basis for this phenomenon remains unclear. The cullin ubiquitin ligases are key regulators of protein degradation that are specifically regulated by covalent modification with the ubiquitin-like molecule NEDD8. Cullins control the turnover of proteins with key tumor suppressive roles in cell cycle regulation, DNA damage responses, and cell death. Accordingly, the inappropriate loss of several NEDD8-cullin regulated targets is known to promote cancer progression and drug resistance suggesting that targeting this pathway may be an effective anticancer strategy. MLN4924 is a novel first-in-class small molecule inhibitor of NEDD8 activating enzyme (NAE); the proximal regulator of NEDD8- mediated degradation. Our preliminary data suggest that NEDD8-mediated protein turnover is required for AML progression as its inhibition with MLN4924 produces stable disease regression in mice with AML. We hypothesize that inhibition of NAE activity will antagonize AML disease progression, disrupt cellular redox status, and increase the efficacy of standard AML therapy. In Aim 1, we will investigate whether oxidative stress induction is an essential mechanism of cell death induced by NAE inhibition. In Aim 2, we will evaluate the role that autophagy plays in NAE inhibition-mediated cell death. Finally, in Aim 3 we will determine the mechanism(s) by which NAE inhibition enhances the activity of the standard of care drug cytarabine. At the conclusion of these studies, we will have significantly expanded our knowledge regarding the role of NEDDylation in AML pathogenesis and will have generated critical new information required to develop novel strategies to optimally target the NEDD8 pathway for the treatment of AML and other forms of cancer.